Theriogenology xxx (2015) 1–10

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Recent advances toward the practical application of embryo transfer in pigs Emilio A. Martinez*, Cristina Cuello, Inmaculada Parrilla, Cristina A. Martinez, Alicia Nohalez, Jose L. Vazquez, Juan M. Vazquez, Jordi Roca, Maria A. Gil Department of Animal Medicine and Surgery, University of Murcia, Murcia, Spain

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 April 2015 Received in revised form 2 June 2015 Accepted 7 June 2015

Porcine embryo transfer (ET) technology has been in demand for decades because of its potential to provide considerable improvements in pig production with important sanitary, economic, and animal welfare benefits. Despite these advantages, the commercial use of ET is practically nonexistent. However, the two main obstacles hindering the commercial use of ET in pigs in the past several decades (i.e., surgical transfer and embryo preservation) have recently been overcome. A technique for nonsurgical deep-uterine (NsDU) ET of nonsedated gilts and sows, which was seemingly an impossible challenge just a few years ago, is a reality today. The improvements in embryo preservation that have been achieved in recent years and the excellent reproductive performance of the recipients after the NsDU-ET technique coupled with short-term and long-term–stored embryos represent essential progress for the international trade of porcine embryos and the practical use of ET by the pig industry. This review focuses, with an emphasis on our own findings, on the recent advances in embryo preservation and NsDU-ET technologies, which are starting to show potential for application under field conditions. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: Embryo storage Nonsurgical embryo transfer Vitrification Porcine Cryopreservation

1. Introduction There is great interest in the use of embryo transfer (ET) in pig production because of its broad applications, including the movement and introduction of new genetic material (i.e., embryos) into a herd with reduced transportation cost, absence of an effect on animal welfare during transport, and minimal risk for disease transmission. Despite these advantages, the commercial use of ET in pigs is currently practically nonexistent. The main reasons for the limited use of ET in this species have been the requirement for surgical procedures to transfer the embryos to recipients and the difficulties in cryopreserving the embryos. However, in the past decade, new

* Corresponding author. Tel.: þ34 868884734; fax: þ34 868887069. E-mail address: [email protected] (E.A. Martinez). 0093-691X/$ – see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2015.06.002

methodologies have been developed that enable successful nonsurgical ET and efficient embryo preservation. Although the first pregnancy in pigs through nonsurgical ET was reported almost 50 years ago [1], nonsurgical ET was considered an impossible technique for many years because of the complex anatomy of the swine genital tract. However, in the 1990s, several nonsurgical techniques for depositing embryos directly into the uterine body were developed, but none of them were sufficiently successful (reviewed in [2,3]). To overcome some of the physiological and practical limitations of nonsurgical uterine body ET, we developed a new procedure for the nonsurgical deep-uterine (NsDU) ET of nonsedated gilts and sows. During the first attempt at NsDU-ET using fresh embryos, an acceptable reproductive performance of the recipients was achieved [4]. With improvements in the procedure, the results were greatly enhanced [5,6], even when fresh embryos cultured for 24 hours were used [7].

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Although it is possible to routinely cryopreserve embryos from several mammalian species, the cryopreservation of pig embryos has largely been limited because of their high sensitivity to chilling injury [8,9]. At present, vitrification is considered the only suitable method for the long-term storage of porcine embryos. The substantial progress achieved over the past 15 years in vitrification protocols has resulted in high postwarming in vitro survival of morulae and blastocysts even without embryo pretreatments, such as delipidation, cytoskeletal stabilization, or centrifugation (hereafter referred to as “untreated embryos”). Moreover, promising farrowing rates and litter sizes have been reported after the surgical and NsDU transfer of vitrified embryos (reviewed in [3]). However, the number of studies is limited, and these studies have involved low numbers of ETs, which may have been due to the effort and high costs required to obtain large numbers of embryos of this species. Although the use of vitrified embryos coupled with NsDU-ET could be essential for the widespread use of porcine ET, other alternatives for embryo preservation (e.g., short-term [24–48 hours] storage of fresh embryos) should also be considered. Here, we provide a brief summary of the current achievements in porcine embryo preservation and describe some basic aspects of NsDU-ET technology. Finally, we discuss several factors that affect the success of NsDU-ET for its practical application. This review will only refer to the in vivo–derived embryos because from a practical and commercial point of view, they are the only embryos with potential short-term application in pig production. 2. Embryo preservation After collection, the embryos must be stored until they are transferred to the recipients. There are several methods of preserving the embryos. In vitro culture can be used as a method for medium- to short-term embryo storage, whereas vitrification is the only efficient method available for the long-term preservation of pig embryos. 2.1. Medium- to short-term storage of embryos A potential method to maintain the developmental capacity of the embryos for brief periods is IVC, which can be used as a method for medium-term (72–120 hours) or short-term (24–48 hours) embryo storage. For medium-term storage, the embryos must be collected at a very early developmental stage, which prevents development beyond the unhatched blastocyst stage at the end of the culture. This issue is essential because embryos must be protected by an intact ZP for sanitary reasons [10]. High blastocyst formation rates from one-, two-, and four-cell embryos cultured in vitro for 72 to 120 hours have been reported [11,12]. Although these blastocysts had lower cell numbers compared with their in vivo counterparts, no difference between the two types of blastocysts was noted in the in vivo developmental ability after surgical ET [13,14]. Because of the limited number of studies, more research is needed to evaluate the effectiveness of different culture media and temperatures

on the in vitro and in vivo development of medium-term– stored embryos. To minimize the detrimental effect of the culture conditions on embryo quality, a shorter culture period, such as 24 hours, can be used. A period of 24 hours between the collection and transfer should be sufficient for the regional, national, and even international transportation of the embryos to their recipients. Despite its importance, research on short-term porcine embryo culture has been limited. Using this storage method, acceptable farrowing rates (50%–60%) and litter sizes (5–8 piglets born) have been obtained after surgical transfers of embryos cultured for 24 to 30 hours [15,16], which indicates that short-term– cultured embryos are able to develop to term. In addition, several types of serum-containing or BSA-containing media and several temperatures have been shown to be effective for short-term embryo culture [17], although the in vivo developmental capacity after the transfer of the cultured embryos was not evaluated. In a recent study, we achieved high reproductive performance in the recipients after NsDU-ET using fresh cultured morulae kept at 37  C for 24 hours in a chemically defined medium [7]. The results of this study indicated that Tyrode’s lactate (TL)-HEPESpolyvinyl alcohol (TL-PVA) provided a chemically defined medium capable of maintaining a high in vitro viability of porcine morulae cultured at 37  C for 24 hours. More than 95% of the embryos cultured under these conditions progressed to the unhatched blastocyst stage during culture (Fig. 1), and, unlike the controls, none of them hatched at the end of the culture, which is, as noted previously, essential for sanitary reasons. Although culture caused certain embryo developmental delays, the resulting blastocysts retained their potential to develop to term in the same manner as uncultured blastocysts (Fig. 2). Interestingly, in that study, for the first time, the embryos could be collected, handled, cultured, and transferred in the defined TL-PVA medium. This fact is essential because embryo culture media usually contain serum or serum components, which carry a risk of disease transmission [18], an important limitation for embryo transport. Currently, we are evaluating the potential to increase the storage period of fresh morulae and blastocysts up to 48 hours by using various culture media and temperatures. Although the in vivo development of these embryos cultured for 48 hours has not yet been fully investigated, the preliminary in vitro results are promising. Moreover, for morulae and early blastocysts, we are evaluating the potential to prolong the storage period to 72 hours after embryo collection because this would enable worldwide embryo transport and transfer. The use of short-term embryo storage in combination with NsDU-ET technology opens new possibilities for the sanitary and safe national and international trade of fresh porcine embryos as well as the practical application of ET in pigs under field conditions. 2.2. Long-term storage of embryos Although a short-term culture period may permit the international transport of embryos, cryopreservation of embryos is preferable. Embryo cryopreservation allows for

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Fig. 1. In vitro development of fresh in vivo–derived morulae (A, C) cultured for 24 hours at 37  C in a defined medium, Tyrode’s lactate and polyvinyl alcohol supplemented with 10-mM HEPES (B), or cultured in North Carolina State University-23 medium supplemented with BSA and fetal calf serum at 38.5  C in humidified air with 5% CO2, controls (D). Note the developmental delay of embryos cultured in the defined medium. Scale bar: 100 mm for all images.

an indefinite storage of embryos, an increased selection pressure in select herds, the rescue of premium genetics from diseased herds, and the international export and import of potential breeding stocks [19]. Fifteen years ago, vitrification was proposed as an alternative to conventional slow-freezing procedures and is, to date, the only suitable procedure for the cryopreservation of porcine morulae and blastocysts. Since the beginning of the porcine embryo vitrification technology [20], many factors that can profoundly affect the survival postwarming rates of embryos have been evaluated. The type of cryodevice used for vitrification is an important factor in improving the effectiveness of embryo vitrification. Various types of devices have been used to hold the embryos during vitrification, storage, and warming. Initially, embryos were vitrified in 0.25-mL straws, which limit the cooling rate to less than 2500  C/min [21]. The success of porcine embryo vitrification was improved by using straws with a smaller inner diameter and wall thickness such as open pulled straws (OPSs) [20] and superOPS [22]. These devices were used to decrease the volume of medium surrounding the embryos, thereby increasing the cooling and warming rates above 20,000  C/min, permitting a reduction in cryoprotectant concentration

[23] and, consequently, a lower toxicity of the vitrification solutions [24]. Subsequently, many different containers, including cryoloops, solid-surface vitrification, microdrops, and cryotops (reviewed in [25]), have been successfully used. However, these devices allow direct contact of the medium containing the embryos with liquid nitrogen, which expose the embryos to potential risk of microbial contamination. To avoid contamination of pathogens, closed systems have also been successfully developed to vitrify in vivo–derived porcine embryos [26]. The stage of embryonic development is another key factor in the success of vitrification because it affects the resistance to chilling injury of pig embryos and, therefore, the postwarming embryo survival [27–29]. The sensitivity of porcine embryos to cryopreservation has been ascribed to their high lipid content in the cytoplasm [9,30], which decreases as the development stage increases. The successful vitrification of porcine embryos has, to date, been limited to the morula and blastocyst stages [24,26,31–34]. Effective methods for the cryopreservation of earlier embryos (zygotes and two- to four-cell embryos) could be valuable for genetic rescue in sanitary crises that involve the extermination of all animals, where it is not possible to choose the collection day to obtain the most suitable

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embryos for cryopreservation, and for the development of new biotechnologies, such as cloning programs [35–37]. However, studies on the cryopreservation of early-stage porcine embryos have been limited and have yielded poor results [11,38,39]. Other factors evaluated for improving cryopreservation include the use of specific cryoprotective agents to assess their toxicity and concentration, cooling and warming rates, the composition of the vitrification and warming solutions, and the use of embryo pretreatments before vitrification (cytoskeletal stabilizing agents, polarization of lipid and chemical delipidation; reviewed in [2,19,40,41]). Because a simple, safe, and standardized vitrification protocol requiring no special embryo pretreatments, which warrants optimal embryo survival and quality, is most suitable for commercial purposes, we developed a chemically defined medium for the vitrification and warming of untreated morulae and blastocysts (Fig. 3) [34]. We also simplified the conventional three-step warming procedure with a direct warming procedure (one-step dilution) [42–44], which is ideal for direct NsDU-ET under field conditions. Postwarming in vitro survival rates of 40% to 70% were initially reported with untreated morulae and blastocysts vitrified using the OPS method (reviewed in [2,40]). With the advancements in vitrification protocols that have been achieved in recent years, our laboratory and others have obtained high postwarming in vitro survival of untreated morulae (>80%) and blastocysts (>90%; Fig. 4; reviewed in [3]), which confirms earlier findings that embryo pretreatments before vitrification are not necessary [32]. 3. Embryo transfer methods

Fig. 2. In vitro and in vivo development of fresh in vivo–derived morulae cultured for 24 hours. Collected morulae (blue bars; 0 hours of culture) were cultured for 24 hours at 37  C in a defined medium (Tyrode’s lactate and polyvinyl alcohol supplemented with 10-mM HEPES; gray bars). Controls were morulae cultured in North Carolina State University-23 medium supplemented with BSA and fetal calf serum at 38.5  C in humidified air with 5% CO2 (black bars). (A) Developmental scores (1, morula; 2, early blastocyst; 3, full blastocyst; 4, expanded blastocyst; and 5, hatching or hatched blastocyst) and total cell numbers. (B) Frequency distribution of the developmental embryonic stage after culture. (C) Farrowing rates and litter sizes after transfer of blastocysts resulting from fresh in vivo–derived morulae cultured in the defined medium (n ¼ 24). Uncultured embryos collected at the blastocyst stage were directly transferred to the recipients within 3 hours of collection (white bars; n ¼ 25). Different letters represent differences (P < 0.001) between groups. (For interpretation of the references to color in this figure, the reader is referred to the Web version of this article.) Data adapted from Martinez et al. [7].

In the 1960s, laparotomy was the sole method used to transfer pig embryos into recipients. However, Polge and Day [1] reported that pregnancy could be established in pigs by using a nonsurgical ET technique. Despite this finding, nonsurgical ET was considered impossible in previous decades. The complex anatomy of the cervix and uterus was the principal obstacle to overcome when inserting a catheter during metaestrus (Fig. 5). In the 1990s, various methods of depositing embryos directly into the uterine body by using nonsurgical techniques were reported, but the reproductive performance of the recipients were generally unsatisfactory (reviewed in [2,45]). Among these procedures, the most promising involved the placement of embryos into the uterine body of nonsedated sows [46]. Using this technique under field conditions, 40% farrowing rates and 7.2 piglets/litter were achieved after the transfers [47]. Although these results can be considered as acceptable, improvements were still needed to increase the reproductive performance of the recipients after nonsurgical ET. Because results from surgical transfers indicated that the uterine body is a less appropriate place for the deposition of the transferred embryo than the middle or anterior quarter of the uterine horn [48] and to overcome other limitations of nonsurgical uterine body ET (e.g., the embryo stage [only blastocysts

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uterine horn in less than 5 minutes. The procedure is well tolerated by the recipients (no reaction in more than 90% of the recipients), and there are no perforations of the uterine wall during insertions. In addition, the use of aseptic protocols during the procedure (by using a clean ET room and sterile instruments, carefully cleansing and disinfecting the perineal area, and administering antibiotic on the day of ET) prevents uterine infections after transfers. Although NsDU-ET is a simple, safe, effective, and practical procedure, transfer skills are improved with practice, thereby leading to an increase in the farrowing rates and a decrease in the duration of the catheter insertions [3]. Fig. 3. In vitro survival (least-squares mean; standard error of the mean [SEM] ¼ 6.7) and hatching rates (least-square means; SEM ¼ 7.2) of fresh in vivo–derived morulae and blastocysts vitrified and warmed in TCM-199– HEPES supplemented with 0.1% polyvinyl alcohol (defined medium) or 20% newborn calf serum (control medium). Data adapted from Sanchez-Osorio et al. [34].

yield acceptable results] and the type of recipient [only possible in sows]), we developed a new nonsurgical procedure to transfer porcine morulae and blastocysts into the depth of uterine horns of nonsedated gilts and sows. 4. Development of the nonsurgical deep-uterine embryo transfer procedure The safety and effectiveness of the NsDU-ET procedure has been recently reviewed [3,49]. Briefly, we developed an instrument, currently produced by Minitube (Tiefenbach, Germany), with the following two properties: the necessary propulsion force to penetrate through the cervical canal and the necessary flexibility to progress through the uterine horn without perforating the uterine wall (Fig. 5). In approximately 90% of gilts and sows, the catheter can be correctly inserted into the second or third quarter of the

5. Factors affecting the success of nonsurgical deeputerine embryo transfer Many factors can affect the reproductive performance of recipients after NsDU-ET. In addition, as with the development of any new technology, it is necessary to reevaluate other specific factors affecting the success rate of NsDU-ET because they were based on the use of surgical transfer or nonsurgical transfer into the uterine body. Some of these factors (e.g., superovulation of the donors and the degree of synchrony between the stage of embryo development and the recipients) coupled with NsDU-ETs have been reviewed in detail elsewhere [3] and are not discussed herein. Thus, we will discuss other factors and their impacts on the practical use of ET in the pig industry in the following.

5.1. Use of fresh and preserved embryos 5.1.1. Fresh and short-term–stored embryos The results achieved after the surgical and nonsurgical transfer of fresh and short-term–stored (24 hours) embryos are summarized in Table 1. Little information is available on the reproductive performance of recipients after surgical transfers of fresh

Fig. 4. Development of in vivo–derived porcine embryos after vitrification and warming. Fresh in vivo–derived compacted morulae before vitrification (A) and immediately after warming (B); evolution of morulae to early and full blastocysts at 24 hours after warming (C) and to expanded and peri-hatching blastocysts at 44 hours after warming (D). Note the presence of a degenerated embryo in (D). Fresh in vivo–derived blastocysts before vitrification (E) and immediately after warming (F); (G, H) evolution of blastocysts at 24 hours (G) and 48 hours (H) after warming. Note the presence of expanded, hatching, and hatched blastocysts in (H). Scale bar: 100 mm for all images.

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Fig. 5. Reproductive tract of a sow during metaestrus (A). Internal wall of the uterine body, cervix, and vagina (B). Note that the cervical folds in the uterine region of the cervix are more closely packed than those in the vaginal portion. Transfer catheter developed for nonsurgical deep-uterine embryo transfer of gilts and sows at Days 4 to 6 of the estrous cycle (C). Silhouette of the transfer catheter into a uterine horn (D). Position of the insemination catheter in the entry of the cervix and a detail of the transfer catheter through the cervical folds (E).

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Table 1 Reproductive performance of the recipients after surgical and nonsurgical transfer of fresh and short-term–stored porcine embryos. Recipients (n)

Embryos transferred (n)

Embryo transfer method

Embryo storage

Pregnancy/farrowing (%)

Fetuses/piglets born

Reference

41 20 16 39 19 21 27 44 16 30 24 46 74 25 12 19 24

11–18 16–23 15–20 17 14 14–21 28–30 17–32 13–55 12–20 24–31 30 30 30 19 19 30

Surgical Surgical Surgical Surgical Surgical NsUB NsUB NsUB NsUB NsUB NsDU NsDU NsDU NsDU Surgical Surgical NsDU

Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh Fresh 24 h 24 h 24 h

75.6a 65.0a 88.0a 79.5/79.5 63.2/63.2 33.3/33.3 59.2a 68.2/40.9 31.2/31.2 16.7a 70.8/70.8 75.1/73.2 85.1/81.1 92.0/92.0 58.3/58.3 63.1/47.4 91.7/91.7

10.2a 9.3a 8.2a 8.1 7.1 6.7 10.9a 7.2 6.2 5.8a 6.9 9.4 9.6 9.4 8.3 5.6 9.0

[50] [13] [48] [51] [52] [46] [53] [47] [54] [55] [4] [5] [6] [7] [15] [16] [7]

Abbreviations: NsDU, nonsurgical deep-uterine embryo transfer; NsUB, nonsurgical uterine body embryo transfer. a Data obtained at Days 25 to 40 of pregnancy.

embryos. Overall, these studies have reported pregnancy rates of 60% to 90% with 8 to 10 fetuses per pregnant recipient at 25 to 40 days of pregnancy [13,48,50] or farrowing rates and litter sizes of 60% to 80% and 7 to 8 piglets born, respectively [51,52]. These results are higher than those reported after nonsurgical ET into the uterine body (farrowing rates of 33%–40%; litter sizes of 6.7–7.4 piglets) [46,47] but similar to those achieved in the first attempt of the NsDU-ET technique (71.4% farrowing rate and 6.9 piglets/litter) [4]. With NsDU-ET procedural improvements, including the use of aseptic protocols during transfers, an adequate degree of estrous synchrony between recipients and donors and an operator training period (reviewed in [3]), the reproductive performance of recipients has been enhanced, achieving farrowing rates of approximately 80% and litter sizes of approximately 9.5 piglets/litter [5–7]. The use of short-term–stored embryos has resulted in variable results depending on the ET procedure used. Thus, although acceptable farrowing rates (50%–60%) and litter sizes (5–8 piglets) have been obtained after the transoceanic transport of the embryos (24–30 hours of IVC) [15,16] using surgical ET, excellent reproductive performance (90% farrowing rates with 9.0 piglets/litter) has been reported after NsDU-ET of fresh morulae cultured for 24 hours in vitro [7]. The different numbers of transferred embryos between these studies (19 and 30 for surgical and NsDU-ET procedures, respectively) or differences in the methodology used (e.g., culture media, embryo development stage, and recipients) may be involved in the variability of the results after ETs. Moreover, it is likely that the reproductive performance of the recipients after NsDU-ETs with this type of embryos varies depending on the facilities in the farm for handling the embryos and performing the transfers. 5.1.2. Long-term–stored embryos As noted previously, the substantial progress achieved in recent years in the vitrification protocols has resulted in high postwarming in vitro survival of untreated morulae and blastocysts. Moreover, promising farrowing rates and

litter sizes have been reported after the surgical transfer of vitrified embryos (reviewed in [3]). However, the number of studies is limited, and they involve low ET numbers, which is most likely due to the need for large numbers of embryos at one particular time. The use of NsDU-ET, instead of surgical ET, coupled with embryo vitrification will be essential for the widespread application of porcine ET. In our preliminary experiments that combined both technologies, we obtained acceptable farrowing rates (40%–50%) and litter sizes (5–10 piglets) [44,56], but the number of recipients was low. Therefore, more research is required to confirm the efficiency of NsDU transfer of vitrified embryos. Recently, we compared the effectiveness of surgical and NsDU transfer procedures using vitrified embryos under field conditions and with a relatively high number of recipients (109 sows) [57]. Additionally, we evaluated the effect of the number of nonsurgically transferred vitrified embryos on the reproductive performance of the recipients. From this study, two main conclusions can be drawn: (1) NsDU-ET may be as efficient as surgical ET with

Fig. 6. Pregnancy rates, farrowing rates, and litter sizes (mean  standard deviation) of recipients after surgical transfers of 30 vitrified embryos (S-30) and nonsurgical deep-uterine embryo transfer of 30 (NsDU-30) or 40 (NsDU-40) vitrified embryos. a,bDifferent letters in the same variable indicate differences (P < 0.004). Data adapted from Martinez et al. [57].

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an appropriate number of embryos per transfer and (2) farrowing rates and litter sizes increase with an increased number of transferred vitrified embryos using the NsDU-ET procedure. Effectively, NsDU-ET with 30 vitrified–warmed embryos resulted in a significant decrease in farrowing rates and litter sizes compared with those in the surgical and NsDUET groups with 30 and 40 vitrified embryos, respectively (Fig. 6), which indicated that more vitrified embryos are necessary for nonsurgical ETs than for surgical ETs. This is not a surprising finding because successful nonsurgical ET requires almost twice the number of fresh embryos recommended for surgical transfers [4,51,53]. A reasonable explanation for the difference may be the site of deposition of the embryo within the uterus, which is related to the ET procedure used. In surgical ET, the embryos are deposited into the tip of a uterine horn [50], whereas in NsDU-ET, the embryos are placed deep (middle or anterior quarter) in a uterine horn [3,4,49]. Under physiological conditions, pig embryos at the four-cell stage pass from the oviduct to the uterine horns on Day 4 (Day 0 ¼ onset of estrus) and develop into morulae and blastocysts. These embryos remain near the tip of the uterine horn until Day 6 or 7 of the cycle, when they progress toward the uterine body [58]. Thus, the transfer of morulae and blastocysts to the tip of a uterine horn might have benefits over embryos transferred to more caudal parts of the uterus, as have been found using surgical transfers [48]. It is likely that the uterine environment in caudal locations is less propitious for embryos during these stages of development, resulting in reduced embryo survival. Thus, the transfer of a higher number of embryos, as in the NsDU-ET group with 40 embryos, might reduce the negative effects of the inadequate uterine environment. More research is required to corroborate this hypothesis. Our results clearly showed that pregnancy rates, farrowing rates, and litter sizes increased with an increase from 30 to 40 vitrified embryos transferred with the NsDU-ET procedure [57]. In contrast to this finding, surgical ET studies evaluating the pregnancy rates and the number of viable fetuses on Day 30 of gestation indicated that 20 vitrified embryos might be the optimal number of embryos for transfer to each recipient and that little benefit is gained from the transfer of more embryos [59]. Although there were important methodological differences between the two studies (e.g., ET procedure, breed of recipients, age of recipients, and method of vitrification), previous evidence supports our results. First, pregnancy rates and the numbers of viable embryos on Day 25 of gestation are notably increased in recipients transferred with 24 fresh embryos than in those transferred with 12 fresh embryos [50,60]. Second, litter sizes and piglet production efficiency (calculated as the ratio of the number of live-born piglets to the number of embryos transferred to all recipients) are more than twice as high for surgical or nonsurgical transfer of 25 to 35 vitrified embryos [26,44,61–63] than when transferring only 20 vitrified embryos [56,64,65]. Similarly, in our study, an increase from 30 to 40 vitrified embryos per NsDU-ET increased the piglet production efficiency by 2.5-fold. Overall, these data suggest that an increase in the number of embryos transferred results in a higher number

of viable fetuses at Day 30 of pregnancy and, subsequently, in an increase in the number of piglets born alive. 5.2. Embryonic stage In a previous study that used NsDU-ETs with 30 fresh embryos per transfer, a similar reproductive performance of the recipients was observed between transfers performed with morulae or blastocysts [6], which indicates that the embryos in the morula stage had similar in vivo potential for development as those in the blastocyst stage. Recently, we evaluated the effects of the embryonic stage (morulae and blastocysts) on the farrowing rates and litter sizes after NsDU-ETs of vitrified embryos [57]. We used a fixed number of 40 vitrified embryos that were transferred immediately after warming and, therefore, without selecting viable embryos (see Fig. 4B, F). For that reason, in each trial, several warmed embryos were randomly designated for IVC to evaluate in vitro development, and the remaining embryos were transferred to the recipients. Although the in vitro survival rates were lower for vitrified morulae (74.7%) than for vitrified blastocysts (90.9%), the embryonic stage, as described for fresh embryos, did not influence the reproductive performance of the recipients. Taking into account these in vitro survival rates, the number of potential viable embryos transferred per recipient included 30 morulae and 36 blastocysts, which is similar to the number used for ETs with fresh embryos [4–7]. These data suggest that the surviving vitrified morulae have similar ability to develop in vivo as the vitrified blastocysts and that the surviving vitrified morulae and blastocysts have a similar ability to develop in vivo compared with fresh morulae and blastocysts. 5.3. Other factors Other factors affecting the success of NsDU-ET technology that must be considered include the genotype and the parity number of the recipients. The choice to use chemically defined medium in each step of ET, the potential for embryo revitrification, and the evaluation of the future reproductive potential of donors and recipients used in ET programs are also important factors for the implementation of the technology. Further research is needed to evaluate these and other factors to improve the safety and efficiency of porcine NsDU-ET. 5.4. Conclusions The technique described in this review is the only nonsurgical procedure to deposit the embryos deep into the uterine horns of nonsedated gilts and sows. The procedure is safe, practical, and well tolerated by the recipients, and it can be performed under field conditions without specific facilities. Important advances have been achieved during the past decade in the development of short-term (24 hours) and long-term (vitrification) storage procedures for fresh morulae and blastocysts. The excellent reproductive performance of the recipients after the NsDU-ET technique coupled with short-term- and longterm–stored embryos represent essential progress for the

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practical use of ET by the pig industry, which will benefit from the numerous applications of this technology, particularly from the exchange of relevant genetic material with a minimal risk of disease transmission. However, because more preserved embryos are necessary for nonsurgical than for surgical ETs to achieve similar reproductive results, greater effort is needed to increase the efficiency of that technology. In addition, although the NsDU-ET catheter can be easily inserted in most of the recipients, further studies are needed to evaluate the influence of recipient age and genotype on the effectiveness of the insertions. Acknowledgments The authors are grateful to Prof. Billy N. Day for his constant help and invaluable advice over the years. The authors also thank the staff of Selección Batalle SA (Girona, Spain), Agropor SA (Murcia, Spain), and Porcisan (Murcia, Spain) for the excellent management of the donors and recipients in the studies reported here. Some of the studies presented in this review were supported in part by MICINN (Ministerio de Ciencia e Innovación)-FEDER (Fondo Europeo de Desarrollo Regional) (Madrid, Spain; AGL2004–07546 and AGL2009– 12091), MINECO (Ministerio de Economía y Competitividad)FEDER (Madrid, Spain; AGL2012-38621), CDTI (Centro para el Desarrollo Tecnológico Industrial) (Madrid, Spain; IDI20140140 and IDI-20140142), and the Fundación Séneca (Murcia, Spain; GERM 04543/07). MINECO is acknowledged for their grant-based support to Cristina A. Martinez and Alicia Nohalez (BES-2013-064087 and BES-2013-064069). References [1] Polge C, Day BN. Pregnancy following non-surgical egg transfer in pigs. Vet Rec 1968;82:712. [2] Cameron RD, Beebe LF, Blackshaw AW. Cryopreservation and transfer of pig embryos. In: Ashworth CJ, Kraeling RR, editors. Control of pig reproduction VII. Nottingham, UK: Nottingham Univ Press; 2006. p. 277–91. [3] Martinez EA, Gil MA, Cuello C, Sanchez-Osorio J, Gomis J, Parrilla I, et al. Current progress in non-surgical embryo transfer with fresh and vitrified/warmed pig embryos. In: Rodriguez-Martinez H, Soede NM, Flowers WN, editors. Control of pig reproduction IX. Leicestershire, UK: Context Products Ltd; 2013. p. 101–12. [4] Martinez EA, Caamaño JN, Gil MA, Rieke A, Mccauley TC, Cantley TC, et al. Successful nonsurgical deep uterine embryo transfer in pigs. Theriogenology 2004;61:137–46. [5] Angel MA, Gil MA, Cuello C, Sanzhez-Osorio J, Gomis J, Parrilla I, et al. The effects of superovulation of donor sows on ovarian response and embryo development after nonsurgical deep-uterine embryo transfer. Theriogenology 2014;81:832–9. [6] Angel MA, Gil MA, Cuello C, Sanzhez-Osorio J, Gomis J, Parrilla I, et al. An earlier uterine environment favors the in vivo development of fresh pig morulae and blastocysts transferred by a nonsurgical deep-uterine method. J Reprod Dev 2014;60:371–6. [7] Martinez EA, Angel MA, Cuello C, Sanchez-Osorio J, Gomis J, Parrilla I, et al. Successful non-surgical deep uterine transfer of porcine morulae after 24 hour culture in a chemically defined medium. PLoS One 2014;9:e104696. [8] Wilmut I. The low temperature preservation of mammalian embryos. J Reprod Fertil 1972;31:513–4. [9] Dobrinsky JR. Cryopreservation of pig embryos. J Reprod Fertil 1997; 52:301–12. [10] Stringfellow DA. Recommendations for the sanitary handling of in vivo derived embryos. In: Stringfellow DA, Seidel SM, editors. Manual of the International Embryo Transfer Society. Savoy, Illinois: IETS; 1998. p. 79–84.

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